Severe congenital neutropenia (SCN) is a group of heterogeneous disorders characterized by severe absolute neutropenia and a maturation arrest of myeloid cells at the promyelocyte stage. Heterozygous mutations in ELANE encoding neutrophil elastase (NE) have been detected in most SCN patients. Expression of the NE mutants in myeloid cells induces premature apoptosis. Patients with SCN are at increased risk of developing acute myeloid leukemia (AML) and progression from SCN to AML is associated with mutations in CSF3R encoding the G-CSF receptor (G-CSFR), which lead to C-terminal truncation of the receptor. The truncated G- CSFRs mediate augmented cell proliferation and survival. It is still poorly understood about the mechanism by which the ELANE mutations cause apoptosis and why CSF3R mutations are specifically associated with SCN. We have observed that co-expression of the wild type (WT) NE accelerates apoptosis induced by the NE mutant and that the C-terminal truncation of the G-CSFR blocks the activation of the Elane promoter in response to G-CSF. Our preliminary data also identified transcription factors Stat5a and Stat5b as potential targets of NE in that ectopic expression of NE inhibits Stat5 protein levels and Stat5-depedent proliferation and survival in hematopoietic cells. We hypothesize that the protease activity of WT NE plays an important role in apoptosis induction by the NE mutants and that the nonsense mutations in CSF3R abolishes NE mutant- induced apoptosis by suppressing the expression of the NE mutants and directly counteracting their pro- apoptotic action. Three specific aims are proposed to test these hypotheses. In aim 1, we will evaluate the involvement of WT NE in apoptosis induction by the NE mutants. We will assess the effects of NE knockdown and overexpression on apoptosis induction by the NE mutants, and determine whether the protease activity of NE is required for apoptosis induced by the NE mutants. In aim 2, we will investigate the effects of the truncated G-CSFR on NE expression and apoptosis induction by the NE mutant in primary human bone marrow cells. In aim 3, we will further explore the role of NE in the negative regulation of Stat5 expression/function and address whether co-expression of NE and the NE mutants display a synergistic inhibitory effect on Stat5-depdent proliferation and survival. The proposed studies may provide critical insight into the pathophysiology of SCN and its progression to AML, and may lead to the development of new therapeutic strategies for the treatment of SCN. PUBLIC HEALTH RELEVANCE: Neutrophil elastase is a protease that is produced at extremely high levels during the early development of neutrophils, a type of white blood cells involved in host defense against bacterial infections. In this proposal, we will investigate how mutations in neutrophil elastase gene cause severe congenital neutropenia (SCN), a condition with an abnormal decrease in the number of neutrophils in the blood, and why patients with SCN are at increased risk for developing leukemia. The proposed studies will help us understand the underlying pathogenic mechanisms of SCN and may lead to the identification of potential targets for therapeutic intervention in the treatment of SCN.